International Journal of
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Comparative screening of
and anti-haemolytic activities from non
Aruna LH
1, Sanjeevkumar CB
*Corresponding author:
Ramesh L Londonkar
1Department of Biotechnology,
Biopharmaceutical & Nanobiotechnology Laboratory, Gulbarga University, Kalaburagi, Karnataka, India.
Received: 09 February 2017 Accepted: 22 June 2017 Published: 02 September 2017
A b s t r a c t
Pterocarpus marsupiummedical ailments. In view of this, marsupium
fo
capacity assessment and
Additionally, RBC’S were also used to explore the potentiality of different extracts to shield RBC in oxidative stress
effective pharmacological activity in all assays when compared with petroleum ether extract and their respective standards. This reveals the use of
source of antioxidant, anti opportunity for further investigation.
for producing such bioactivities is under process. Key
haemolytic activity, petroleum ether extract, chloroform e
Introduction
Free radicals are regarded as fundamental biochemistry which forms an important part of aerobic metabolism. They are also important for maintaining normal physiological functions
signaling molecules, regulation of signal transduction and gene expression, activation of receptor and nuclear transduction Imbalance of this metabolism due to metabolic and other environmental factors generates excessive ROS that give rise to oxidative stress. This results in the damage of various biomolecules like DNA, lipids and proteins which is affiliated with several illnesses like cancer, neurodegenerative diseases, atherosclerosis, infertility, hepatitis and rheumatoid arthritis [2]. It also results in an inflammatory pathological state characterized by increased expression of pro-inflammatory mediators, chemokines i.e alpha, cytokines, recruitment of adhesion molecules and caspases which are the crucial factor in many human diseases overcome these hazards, human body has their own antioxidant defense mechanism that is synthesized within the body or taken in diet form. Accordingly, antioxidants act as free-radical scavengers by safeguarding living entity from destruction generated by
DOI:10.5138/09750185.2021
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and redistribution provided that the original author and source are credited.
International Journal of Phytomedicine 9 (2017) 39
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Original Research Article
Comparative screening of
in-vitro
free radical scavenging, anti
haemolytic activities from non-polar solvent extracts of
marsupium
, Sanjeevkumar CB
1, Amarvani PK
1, Ramesh L Londonkar
A b s t r a c t
Pterocarpus marsupium is widely used as ‘Rasayana’ in ayurvedic system for curing several medical ailments. In view of this, petroleum ether and chloroform extracts of
marsupium bark was scrutinized to unfold free radical scavenging by measuring their capability for scavenging DPPH radical, ABTS, phosphomolybdenum assay as well as reducing power capacity assessment and anti- inflammatory activity by inhibition of protein denaturation. Additionally, RBC’S were also used to explore the potentiality of
different extracts to shield RBC in oxidative stress condition. Chloroform extract showed an effective pharmacological activity in all assays when compared with petroleum ether extract and their respective standards. This reveals the use of Pterocarp
source of antioxidant, anti-inflammatory, anti-haemolytic agent
opportunity for further investigation. Isolation of active compounds from this plant responsible for producing such bioactivities is under process.
Keywords: Pterocarpus marsupium, antioxidant activity, anti-inflammatory activity, anti haemolytic activity, petroleum ether extract, chloroform extract
Free radicals are regarded as fundamental biochemistry which forms an important part of aerobic metabolism. They are also maintaining normal physiological functions such as signaling molecules, regulation of signal transduction and gene , activation of receptor and nuclear transduction [1]. Imbalance of this metabolism due to metabolic and other environmental factors generates excessive ROS that give rise to This results in the damage of various biomolecules like DNA, lipids and proteins which is affiliated with several lnesses like cancer, neurodegenerative diseases, atherosclerosis, . It also results in an inflammatory pathological state characterized by increased inflammatory mediators, chemokines i.e.TNF-alpha, cytokines, recruitment of adhesion molecules and caspases which are the crucial factor in many human diseases [3].To overcome these hazards, human body has their own antioxidant defense mechanism that is synthesized within the body or taken in radical scavengers by safeguarding living entity from destruction generated by
abnormal ROS production. In recent years, the quest for natural antioxidants is gaining importance in food processing and pharmaceutical industry due to their presumed safety, nutritional and therapeutic values that restitute unnatural antioxidants which are restrained for their carcinogenicity
medicinal plants has implied antioxidant presence such as ta phenolics, flavonoids and its consumption reduced morbidity, mortality caused from degenerative disorders
these biological properties, plants have reconnoitred for anti hemolytic activity to cure anemia
found in ample amount in an individual, where oxidation reduces membrane protein components and disrupts micro
lead to their lysis, liberating haemoglobin, which can be quantified by a spectrophotometer [6]. Hence, substantial attentio
focused towards credentials of plants with antioxidant, anti inflammatory and anti-haemolytic abilities.
Pterocarpus marsupium is a huge tree, generally known as Vijayasar or Indian Kino. It is belonging to family Fabaceae, grown in India, Nepal and Srilanka. The heartwood of this plant
as an anti-inflammatory ,anti-helmintic, astringent and treating diarrhoea, skin diseases, diabetes, asthma, bronchitis etc Different pieces of bark are used as antacid, anti
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
399-406
free radical scavenging, anti-inflammatory
polar solvent extracts of
Pterocarpus
Londonkar
1*
is widely used as ‘Rasayana’ in ayurvedic system for curing several petroleum ether and chloroform extracts of Pterocarpus was scrutinized to unfold free radical scavenging by measuring their capability r scavenging DPPH radical, ABTS, phosphomolybdenum assay as well as reducing power inflammatory activity by inhibition of protein denaturation. Additionally, RBC’S were also used to explore the potentiality of Pterocarpus marsupium . Chloroform extract showed an effective pharmacological activity in all assays when compared with petroleum ether extract Pterocarpus marsupium as a potent and would provide an ample Isolation of active compounds from this plant responsible
inflammatory activity,
anti-abnormal ROS production. In recent years, the quest for natural antioxidants is gaining importance in food processing and ceutical industry due to their presumed safety, nutritional and therapeutic values that restitute unnatural antioxidants which are restrained for their carcinogenicity [4]. Scientific research on medicinal plants has implied antioxidant presence such as tannins, phenolics, flavonoids and its consumption reduced morbidity, mortality caused from degenerative disorders [5]. In addition to these biological properties, plants have reconnoitred for anti-hemolytic activity to cure anemia-related disorders. RBC’s are found in ample amount in an individual, where oxidation reduces membrane protein components and disrupts micro-constituents that lead to their lysis, liberating haemoglobin, which can be quantified . Hence, substantial attention has been focused towards credentials of plants with antioxidant,
anti-haemolytic abilities.
is a huge tree, generally known as Vijayasar or Indian Kino. It is belonging to family Fabaceae, grown heartwood of this plant are used helmintic, astringent and treating diarrhoea, skin diseases, diabetes, asthma, bronchitis etc [7]. Different pieces of bark are used as antacid, anti-diarrheal, for
ISSN: 0975-0185
PAGE | 400 |
treatment of toothache and diabetes [8]. A collection of flavonoids,their derivatives is isolated from various portion of plant and are also a opulent source of polyphenolic compounds [9].Thus, considering its traditional claims, chemical constituents and pharmacological activities, the current research work is designed.
Materials and Methods
Chemicals
Chemicals such as ABTS (2, 20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), DPPH (1, 1 diphenyl-2-picrylhydrazyl), potassium persulfate, potassium ferricyanide and aluminium chloride were purchased from Sigma Chemicals (Steinheim,Germany).Butylated hydroxytoluene (BHT),ascorbic acid and diclofenac sodium were purchased from HiMedia (India). All chemicals and solvents used in the study were of analytical grade.
Plant material
Pterocarpus marsupium Roxb.bark was collected from Kalaburagi district, Karnataka, in midst of June, 2015. It was identified and authenticated by the Department of Botany, Gulbarga University, Kalaburagi, Karnataka.
Preparation of plant extract
Freshly collected bark of Pterocarpus marsupium Roxb. was washed beneath running faucet water and later through distilled water, shade dried and then powdered to required particle size. The bark powder (100g) was successively extracted by Hot Soxhlet extraction with non polar solvents like petroleum ether and chloroform. The extract was heated at 30-40ºC in hot air oven till the solvent got evaporated. Dried extract was kept in refrigerator at 4ºC for future use.
Antioxidant activity
The antioxidant activity of non polar solvent extracts was determined by different in vitro assays such as DPPH, phosphomolybdenum, ABTS and reducing power. The experiments were carried out in triplicates.
DPPH radical scavenging activity
The free radical scavenging activity of bark extracts against stable DPPH was performed using the method [10]. Briefly, 0.004%w/v of DPPH radical solution was prepared in methanol; 900μl of this solution was mixed with varying concentration of extracts or standard ascorbic acid solution and kept in dark place for 30
minutes. The absorbance was measured at 517nm. Scavenging capacity of DPPH radicals (%Inhibition) was measured by the following formula and finally calculated 50% inhibition concentration (IC50) from the graph of inhibition percentage plotted against extract concentration.
DPPH radical scavenging activity (%) = (Ac − As)/Ac × 100,
Where, Ac is the absorbance of control and As is the absorbance of test samples.
Phosphomolybdenum assay
The antioxidant activity of samples was evaluated by the green phosphomolybdenum complex according to the method of Prieto et al [11]. Different concentrations (5-100µg/ml) of extracts are combined with 1mL of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate) and incubated in a boiling water bath at 95˚C for 90 minutes. After incubation, these tubes were normalized to room temperature for 20 minutes and the absorbance of the reaction mixture was measured at 695nm against a blank using Ascorbic acid and BHT as standard reference.
ABTS radical scavenging activity
ABTS radical cation decolorization activity was determined by the method of Izabela et al [12]with a slight modification. ABTS and potassium persulfate were dissolved in distilled water to a final concentration of 7mM and 2.45mM respectively. These two solutions were mixed and the mixture was allowed to stand in the dark at room temperature for 16h before use in order to produce ABTS radical (ABTS•+). The ABTS radical solution was diluted with ethanol to give an absorbance of 0.700±0.02 at 734nm. Different concentrations (5-100µg/ml) of non polar extracts were added to diluted ABTS radical cation solution (1ml) and the absorbance reading was taken 6 minutes after mixing using the spec-trophotometer at 734nm. The ABTS scavenging capacity of the extract was compared with that of BHT and ascorbic acid and percentage inhibition calculated as ABTS radical scavenging activity.
ABTS radical scavenging activity % = (Ac − As)/Ac × 100,
Where, Ac is the absorbance of ABTS mixture and As is the absorbance of ABTS mixture with test samples.
Reducing power assay
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of solution (0.5ml) was mixed with distilled water (0.5ml) and afreshly prepared ferric chloride solution (0.1ml). The absorbance was measured at 700nm. Control was prepared in similar manner excluding samples. Ascorbic acid, BHT at various concentrations was used as standard.
Anti-inflammatory activity
Protein denaturation method
The inhibition of albumin denaturation was assessed according to the modified method of Asha et al [14].The reaction mixture (5mL) consisted of 0.2mL of egg albumin (from fresh hen’s egg), 2.8mL of phosphate buffered saline (PBS, pH 6.4) and 2mL of varying concentrations of the different extracts (final concentrations 62.5, 125, 250, 500, and 1000 µg/mL).The mixtures were incubated at 37°C for 15min and then heated at 70°C for 5min. After cooling, their absorbance was measured at 660 nm (UV-1800 spectrophotometer, Shimadzu), with double distilled water used as a blank. The diclofenac sodium was used as reference control. The turbidity was determined using UV-VIS spectrophotometer at 660 nm. The percentage inhibition of protein denaturation was determined by the formula and IC
50 value was calculated. % Inhibition = (Vc -Vt/ Vc) × 100,
where Vt = absorbance of the test sample, Vc = absorbance of positive control.
Anti-haemolytic activity
Preparation of human erythrocyte suspension and
haemolysis assay
The human blood sample (5-10ml) were obtained from healthy volunteers in sterile Alsever’s solution and centrifuged at 3000 rpm for 15minutes. The packed cells were washed with sterile Phosphate Buffer Saline (PBS) 3times and 10% erythrocyte suspension was prepared for haemolytic study. The haemolytic activity of the crude extract was tested under in vitro conditions [15]. To 0.5ml of freshly prepared human erythrocyte suspension, various concentrations (62.5–1000μg/ml) of extract were added with 2ml hyposaline solution. After 30 minutes incubation at room
temperature, cells were centrifuged and the supernatant was used to measure the absorbance of the liberated haemoglobin at 540nm. Two controls were prepared without extracts; negative control received sterile phosphate buffer saline, while positive control received hyposaline. The average value was calculated from triplicate assays. Haemolysis percentage for each sample was calculated by the following equation
Percentage prevention of haemolysis = Absorbance of treated sample × 100
Absorbance of control
Statistical analysis
Data was expressed as mean ± SD. Analyses of variance (ANOVA) were done for the comparison of results using Fischer's test. Statistical significance was set at p˂0.05.
Results and Discussion
Antioxidant activity
DPPH radical scavenging activity
DPPH assay mainly depends on the hydrogen donating capacity to scavenge DPPH radicals. Antioxidants respond to the DPPH by reducing the DPPH molecules which is equivalent to their OH−ions prevailing. The reduced capacity of the DPPH radical, when a DPPH electron binds to a radical scavenger is estimated in colour change from purple to yellow suggesting that the decrease of absorbance by antioxidants action is proportional to the number of residual DPPH [16].The free radical scavenging abilities of chloroform extract (73.77%) was increased in a concentration-dependent manner followed by petroleum ether extract (50.73%) compared with standards ascorbic acid(96.14%) and BHT(89.81%) is plotted in Figure-1.These findings were comparable to the previously reported chloroform extracts of Sida acuta that showed high inhibition at low concentration compared with standard reference [17].Further, DPPH result was also expressed as IC50 (half maximal inhibitory concentration) value and lower value indicates better antioxidant capacity as presented in Table-1.
Table-1. IC50 values of DPPH, ABTS and absorbance of Phosphomolybdenum, Reducing Power of ascorbic acid, BHT, petroleum ether and chloroform extracts of Pterocarpus marsupium bark
Samples DPPH
(μg/mL)
ABTS (μg/mL)
Phosphomolybdenum (Abs. at 695 nm)
Reducing power (Abs. at 700 nm) Ascorbic acid 3±0.081 3±0.047 0.37±0.004 1.02±0.008
BHT 3.5±0.14 4±0.12 0.29±0.016 0.56±0.020
PAGE | 402 |
Results represented in means ± standard deviation (n = 3)Figure-1.DPPH radical scavenging activities of ascorbic acid, BHT, petroleum ether and chloroform extracts. Values are Means ± SD (n = 3)
Phosphomolybdenum assay
This method is based on the reduction of phosphomolybdic acid to phosphomolybdenum blue complex by sodium sulfide. The obtained phosphomolybdenum blue complex is oxidized by the addition of nitrite and this causes a reduction in intensity of the blue colour. The antioxidant capacity of petroleum ether extract,
chloroform extract, ascorbic acid and BHT are presented in Figure-2 and Table-1, which certainly indicates dose dependent curve. The chloroform extract of Withania somnifera has showed maximum antioxidant capacity in term of ascorbic acid equivalent with maximum phenol content that is in similarity with our results [18].
Figure-2.Phosphomolybdenum assay of ascorbic acid, BHT, petroleum ether and chloroform extracts. Values are Means ± SD (n = 3)
ABTS radical scavenging activity
ABTS (2-2՝azinobis [3-ethylbenzthiazoline-6-sulphonic acid]) assay is a powerful assay used to determine the chain-breaking antioxidants in case of lipid peroxidation and antioxidant activity of hydrogen donating antioxidants. This assay involves the oxidation of ABTS to form an intensely-green colored nitrogen-centered ABTS•+ [19]. As showed in Figure-3, the chloroform extract of
Pterocarpus marsupium exhibited the highest antioxidant activity (83.80%) whereas, petroleum ether extract of the plant has showed the lowest antioxidant activity (73.83%) clearly indicating chloroform extract acted as a potent antioxidant agent when compared with standards ascorbic acid (88.89%) and BHT (82.27%). A similar result was observed in the chloroform extracts of Bryonopsis laciniosa where it exhibited a highest antioxidant activity followed by ascorbic acid and BHT (90.45%, 68.83% and 0
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55.05%, respectively) [20]. The IC50 values of each sample are presented in Table-1.Figure-3.ABTS radical scavenging activities of ascorbic acid, BHT, petroleum ether and chloroform extracts. Values are Means ± SD (n = 3)
Reducing power assay
Reducing power assay involves the reduction of Fe3+/ferricyanide complex to the Fe2+/ferrous that can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Increased absorbance of the reaction mixture indicates increase in reducing power [21]. As depicted in Figure-4 and Table-1, the chloroform extract had shown good reducing power than petroleum ether
extract that was comparable with standard ascorbic acid and BHT which indicates the presence of reductants or antioxidants in extract which may be responsible for exerting its reducing power.These results are in agreement with the previous study where chloroform extract of Thysanolaena maxima revealed the reductive capabilities compared to ascorbic acid which had the highest reductive activity than the other extracts [22].
Figure-4.Reducing powers of petroleum ether extract, chloroform extract, BHT and ascorbic acid. Values are Means ± SD (n = 3)
Anti-inflammatory activity
Protein denaturation method
Denaturation of proteins is a well-documented cause of inflammation and arthritis, because of auto antigen production.
Numerous non-steroidal anti-inflammatory drugs (NSAIDs) exhibited dose dependent ability to inhibit thermally induced protein denaturation, but the ability of plant material to bring down the protein denaturation could be more effective against inflammation
diseases [13]. Hence, the in vitro anti-inflammatory effect of P.marsupium extracts were evaluated for its ability to prevent denaturation of egg albumin. As shown in Figure-5, petroleum ether and chloroform extracts have showed significant inhibition of egg albumin denaturation in a concentration dependent manner when compared with control diclofenac sodium. Further, petroleum ether and chloroform extracts have showed an IC50 value of 485±0.81µg /mL,150±0.94µg/mL respectively, whereas that of diclofenac sodium was found to be 195±1.24µg/mL. Therefore, it can be stated that the P.marsupium extracts are capable of controlling auto antigen production and thereby show remarkable anti-
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inflammatory activity against inhibition of protein denaturation in vitroFigure-5. In vitro anti-inflammatory activity of petroleum ether and chloroform extracts comparison with Standard (Diclofenac sodium). Values are means ± SD (n = 3).
Anti-haemolytic activity
Preparation of human erythrocyte suspension and
haemolysis assay
RBC haemolysis is a sensitive method for evaluating antihaemolytic properties of medicinal plants. Exposure of HRBC to injurious substances such as hypotonic medium, results in the
membrane lysis accompanied by haemolysis, rendering the cell more susceptible to secondary damage through free radical-induced lipid peroxidation [24]. In this study, chloroform extract significantly stabilized red blood cell membrane, an indication of the extracts ability to prevent haemolysis in hypotonic-stress induced condition when compared with petroleum ether extract and standard ascorbic acid as shown in Figure-6. Membrane stabilizing profiles of various extracts of Lantana camara on bovine red blood cell exposed to both heat and hypotonic induced lyses were reported previously [25].
Figure-6. Percentage prevention of haemolysis of ascorbic acid, petroleum ether and chloroform extracts of Pterocarpus marsupium. Values are means ± SD (n = 3).
Conclusion
Based on the above investigation, Pterocarpus marsupium extracts exhibited significant antioxidant and anti-inflammatory properties,0 20 40 60 80 100
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besides safeguarding against RBC oxidative damage whichcontributed for the presence of phenolic and flavonoid compounds.
Thus this study suggests that the extracts could be of great importance in therapeutic drugs for the implications of human health. Hence, further work is under progress to isolate and elucidate the bioactive principles and to evaluate their mechanisms of action of specific bioactivities.
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